The present invention relates to non-human animals and in vivo methods for testing the efficacy of antibodies directed to antigens expressed by tumors in such animals. In particular, the invention relates to an animal deficient in the expression of one or more Fc receptors. Additionally, such an animal is also immunodeficient, and thus permits the growth of a xenogeneic tumor implant. More specifically, an animal of the invention can be implanted with a human tumor and treated with an antibody directed to an antigen expressed by the tumor, and the anti-tumor efficacy of the antibody is compared between animals with or without Fc receptor expression. The present invention also relates to methods of evaluating the enhanced ability of an existing antibody or Fc-modified antibody to act as an immunotherapeutic to eradicate tumor cells or infectious agents.
Effective immunity against cancer requires the specific recognition and elimination of malignant cells expressing targeted antigens. Antigens recognized on neoplastic cells include viral proteins, products of altered or mutated genes, developmentally reactivated silent gene products, and differentiation antigens expressed by tumor cells and their normal cell counterparts (Houghton, A. N., J. Exp. Med. 180:1 (1994); Jaffee, E. M. and Perdoll, D. M., Curr. Opin. Immunol. 8:622 (1996)). Much of the current effort of vaccine strategies is aimed at eliciting cytolytic T cell responses in which antigen recognition and cytotoxicity are functions shared by a single cell. In antibody-mediated cytotoxicity, however, antigen recognition and cytotoxicity mechanisms are functional properties of distinct cell types.
Therapeutic approaches to generate antigen-specific immune responses against tumors have included both passive immunization with monoclonal antibodies (mAbs) or adoptively transferred activated immune cells, and active immunization using antigens or genes expressing antigens. Passive immunity with antibodies could mediate its cytotoxic effects through complement activation or Fc receptor (FcR) engagement, and immunization with tumor antigens could elicit both cytolytic T cell responses and antibodies capable of triggering effector mechanisms.
Three classes of Murine FcRs for IgG1, IgG2a, and IgG2b have been characterized: the high-affinity receptor Fcxcex3RI and the two low affinity receptors Fcxcex3RII and Fcxcex3RIII (Ravetch, J. V., Curr. Opin. Immunol. 9:121 (1997)). A similar distribution exists in humans, where two genes encode Fcxcex3RIII, A and B, and three genes encode Fcxcex3RII, A, B and C (Ravetch, J. V., Curr. Opin. Immunol. 9:121(1997)). Human Fcxcex3RIIA and IIC are activating receptors, while IIB, as in mice, is an inhibitory FcR. Fcxcex3RI and III are heterooligomeric receptors, requiring co-expression of the common xcex3 chain for their assembly and signaling functions. Cross-linking these receptors results in cell activation. Fcxcex3RIIB, in contrast, is a single chain inhibitory receptor, aborting activation through immune receptor tyrosine-based activation motif (ITAM)-containing receptors. In addition, a distinct Fc receptor for mouse IgG3 has been described (Diamond, B. and Yelton, D. E., J. Exp. Med. 153:514 (1981); Yuan, R. et al., J. Exp. Med., 187:641-648 (1998)). Mice containing genetic disruptions of the xcex3 chain do not express either Fcxcex3RI or III and exhibit functionally impaired antibody-mediated responses, including loss of natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), macrophage phagocytosis, and mast cell degranulation in response to FcR cross-linking (Takai, T. et al., Cell 76:519 (1994)). Furthermore, xcex3 chain deficiency ameliorates the pathogenesis of cytotoxic antibody in models of autoimmune hemolytic anemia and thrombocytopenia (Clynes, R. et al., J. Exp. Med. 184:2385 (1996); Clynes, R. and Ravetch, J. V., Immunity 3:21 (1995)) and the inflammatory cascade initiated by immune complexes in the Arthus reaction (Clynes, R. et al. (1996), supra; Sylvestre, D. L. and Ravetch, J. V., Science 265:1095 (1994)) and autoimmune glomerulonephritis (Clynes, R. et al., Science, 279:1052-1054 (1998); Suzuki, Y. et al., Kidney Int. 54:1166-1174 (1998)). Similar results were observed in Fcxcex3RIII 2- animals, Hazenbos, W. L., et al., 1996, Immunity 5: 181-188. Conversely, animals deficient in the inhibitory FcR, Fcxcex3RIIB, exhibit enhanced inflammatory responses to IgG antibodies or IgG immune complexes (Takai et al., Nature 379:346-349 (1996); Clynes et al., J. Exp. Med. 184:2385 (1996); Suzuki et al., Kidney Int. 54:1166 (1998)). However, selection of antibodies for use as therapeutics has focused on in vitro assays for growth inhibition, complement activation or other effector responses. These assays have not considered the in vivo activities which may be mediated by FcR dependent pathways.
The present invention relates to animals deficient in both immune function and FcR expression or function. Such animals permit the growth of a xenogeneic tumor implant for testing the efficacy of an anti-tumor antibody in vivo. In addition, the mouse FcR may be replaced by its human counterpart. The invention also relates to methods of using such animals in selecting antibodies that mediate anti-tumor ADCC through Fc receptor binding to effector cells. The present invention also relates to methods of evaluating the enhanced ability of an existing antibody or an Fc-modified antibody to act as an immunotherapeutic to eradicate tumor cells or infectious agents.
The invention is based, in part, on the discovery that successful active immunization with a tumor antigen and passive immunotherapy with an anti-tumor antibody in a mouse melanoma model both require the expression of FcR in the tumor-bearing host. In other words, FcR-expressing effector cells are needed to mediate optimal anti-tumor immune responses. Absence of the activating Fcxcex3R reduces antibody efficacy, while the absence of the inhibitory Fcxcex3R enhances antibody efficacy. In addition, both murine and humanized anti-tumor antibodies are effective in causing regression of a human tumor implants in nude mice, whereas the antibodies display much less effective anti-tumor activities in the same animals that are also deficient in activating FcR expression, while displaying enhanced anti-tumor activity in mice deficient in inhibitory FcR expression.
It is an object of the invention to construct a non-human immunodeficient animal which is also deficient in FcR expression or FcR function.
It is also an object of the invention to construct a non-human immunodeficient animal which expresses human FcR instead of its native FcR.
It is another object of the invention to use the aforementioned animals to select an anti-tumor antibody that mediates superior anti-tumor activities in an animal that expresses FcR as compared to an animal that lacks FcR expression. More specifically, the invention relates to a method for selecting an anti-tumor antibody comprising: (a) administering an antibody to a first non-human immunodeficient animal which is implanted with a human tumor; (b) administering said antibody to a second non-human immunodeficient animal which is implanted with said human tumor and said second animal is also deficient in Fc receptor expression or Fc receptor function; and (c) determining the ability of said antibody to retard tumor implant growth in the animal of step (a) as compared to the animal of step (b).
The present invention also relates to a method for selecting an anti-tumor antibody comprising: (a) administering an antibody to a first non-human immunodeficient animal, which is implanted with a human tumor; (b) comparing anti-tumor activity of antibody in step (a) to anti-tumor activity of antibody administered to a second non-human immunodeficient animal which is implanted with said human tumor and second animal expresses human Fc receptor or human Fc receptor function in place of murine receptors; and (c) determining the ability of said antibody to retard tumor implant growth in the animal of step (a) as compared to the animal of step (b).
It is yet another object of the invention to use a non-human FcR deficient animal to select an antibody against an infectious agent. More specifically, the invention relates to a method for selecting an anti-infectious disease agent antibody comprising: (a) administering an antibody to a first non-human animal which is inoculated with an infectious agent; (b) administering an antibody to a second non-human animal which is inoculated with said infections agent and said second animal is deficient in Fc receptor expression or Fc receptor function; and (c) determining the ability of said antibody to inhibit an activity of said infectious agent in the animal of step (a) as compared to the animal of step (b).